observations clinical and immunofluorescent diagnosis

BRITISH MEDICAL JOURNAL 3 APRIL 1971

PAPERS AND ORIGINALS

Observations on Clinical and Immunofluorescent Diagnosisof Parainfluenza Virus Infections

P. S. GARDNER, J. McQUILLIN, R. McGUCKIN, R. K. DITCHBURN

British Medical J'ournal, 1971, 2, 7-12

Summary

Immunofluorescent techniques have been applied tonasopharyngeal secretions for the rapid diagnosis ofparainfluenza virus types 1, 2, and 3 infections. Seventy-fiveinfections were found by isolation techniques; 55 of these hadnasopharyngeal secretions taken and 53 were positive bydirect examination. A comparison of the results of 60 neutral-ization tests with immunofluorescence applied to monkey kid-ney isolations showed complete agreement. Immunofluores-cence appeared to be a satisfactory method for differentiatingthe various haemadsorption viruses. The importance ofparainfluenza viruses and respiratory syncytial virus in croupwas noted and the association of the parainfluenza viruseswith acute respiratory virus infection was confirmed. Theclinical relationship between respiratory syncytial virus andparainfluenza virus type 3 is discussed.

Introduction

Since the first description of the parainfluenza virus group byChanock et al. (1958, 1963) it has become evident that thisgroup of viruses is a major cause of acute respiratory illnessin the young, both for those admitted to hospital (Holzel etal., 1963, 1965) and for those at home (Banatvala et al., 1964),and in this country it is second in importance to respiratorysyncytial (R.S.) virus. Over the past eight months there hasbeen an increase in the occurrence of parainfluenza virustypes 1, 2, and 3 in the Newcastle area, and it was consideredopportune to re-examine both the clinical and the laboratory

Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LPP. S. GARDNER, M.D., DIP.BACr., Consultant Virologist and Honorary

Reader, Department of VirologyJ. McQUILLIN, B.SC., F.I.M.L.T., Senior Scientific Officer, Department

of VirologyR. McGUCKIN, F.I.M.L.T., Chief Technician, Department of VirologyR. K. DITCHBURN, M.B., D.C.H., Senior Research Associate, Depart-

ments of Child Health and Virology

aspects of infection with this virus. The cases described werehospital admissions and showed a range of clinical syndromesvarying from upper respiratory tract infection and croup tobronchiolitis and pneumonia. There was one death.The diagnosis of parainfluenza virus infection has

previously depended on the haemadsorption technique (Vogeland Shelokov, 1957) performed on monkey kidney cells, withconfirmation of a virus isolate by either the haemadsorptionneutralization or the haemadsorption inhibition test. Duringthis study we introduced immunofluorescent techniques forthe identification of parainfluenza virus types 1, 2, and 3,both for the direct examination of respiratory tract materialand for the specific identification of these viruses on tissueculture. To date parainfluenza virus types 4a and 4b have notoccurred in Newcastle, .therefore immunofluorescent tech-niques for their identification have not yet been introduced.This communication falls into two parts: laboratory details

and certain clinical and epidemiological aspects.

Materials and Methods

For a number of years all infants and children admitted withacute respiratory infections to Newcastle hospitals have beeninvestigated for respiratory virus infection. This communi-cation covers the period from February 1968 to November1970. The clinical categories used and the precise method ofobtaining both nasopharyngeal secretions and cough/nasalswabs have been described (Gardner et al., 1960; Gardner,1968; Sturdy et al., 1969), and the immunofluorescent tech-niques are also identical with those used in our earlier studies(Gardner and McQuillin, 1968; McQuillin and Gardner, 1968;McQuillin et al., 1970). During the period when the occur-rence of parainfluenza virus infections were most frequent,between September 1969 and November 1970, all specimenssuitable for examination by immunofluorescence were inves-tigated for parainfluenza virus types 1, 2, and 3 when appro-priate.

Reagents used for Immunofluorescence.-The reagentsused were identical with those previously described for R. S.virus and influenza virus types A and B (Gardner and

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McQuillin, 1968; McQuillin and Gardner, 1968; McQuillin etal., 1970) except for the specific antisera for the parainfluenzaviruses.

The preparation of these antisera was as follows:

Parainfluenza Virus Type 1.-A strain of Sendai virus wasobtained from Dr. A. J. Beale (Glaxo Laboratories). The viruswas grown in the allantoic cavity of fertile hers' eggs, and pooledallantoic fluid with a haemagglutinating titre of 1/1,024, whichhad been clarified by centrifugation, was used for rabbit inocula-tion. Human type 1 virus (Colindale) was not used because it didnot grow on a continuous line of rhesus monkey kidney cells(LLC-MK2) or on fertile hen's eggs. It grew to high titre onrhesus monkey kidney cells but many batches of these containedsimian viruses. The use of primary rhesus monkey kidney cellswould also have created difficulties in absorbing the antiserumwith this cell line. Moreover, preliminary experiments had Shownthat antiserum prepared against Sendai virus was equally effectivein detecting both Sendai and human parainfluenza type 1 virusesin monkey kidney tissue culture.

Parainfluenza Virus Types 2 and 3.-Standard strains ofparainfluenza virus types 2 and 3 were obtained from the Stan-dards Laboratory, Central Public Health Laboratory, Colindale.They were inoculated into LLC-MK2 cells obtained from FlowLaboratories Ltd. Both viruses produced a pronounced cytopathiceffect on these cells. They were passaged several times to producea virus of high titre between 106 and 107. Degenerate cells werescraped off into maintenance medium and disintegrated ultrason-ically. Cell debris was removed by centrifugation at 10,000 r.p.m.for 30 minutes and the supernatant used as the rabbit inoculum.

Rabbits were inoculated three times at seven-day intervals.On the first occasion 1 ml of virus was inoculated intra-venously, 2 ml intraperitoneally, and 1 ml of virus, togetherwith Freund's complete adjuvant, subcutaneously. On the twosubsequent occasions the virus was inoculated intravenouslyand intraperitoneally, the final inoculum being 4 ml and 5 mlrespectively. The rabbits' serum was tested for the presenceof the appropriate parainfluenza virus antibody on the sev-enth day after the final inoculation. If the immunofluorescenttitre of antibody tested in secondary rhesus monkey kidneycells infected with parainfluenza virus was satisfactory(> 1 : 80) the rabbit was exsanguinated.

All three antisera were absorbed twice with LLC-MK2,Bristol, HeLa, and HEp 2 cells to remove all non-specificactivity against monkey kidney and human cells. They werethen titrated by immunofluorescence on rhesus monkey kid-ney cells infected with the appropriate virus, and the highestdilution showing maximum fluorescence was taken as theoptimum dilution. The sera were also titrated on uninfectedrhesus monkey kidney and human cells to ascertain that allnon-specific activity had been removed. In this way anpptimum dilution for immunofluorescent studies wasobtained. These antisera were then tested at their optimumdilutions for non-specific activity, either in tissue culture ornasopharyngeal secretions, against the other twoparainfluenza viruses, R.S. virus, influenza viruses A and B,mumps virus, adenovirus, SV5, and various types of Cox-sackievirus and echovirus. None of the three antisera gaveany cross-reaction against the other two types ofparainfluenza viruses in monkey kidney tissue culture, orwith the other viruses against which they were tested. Toensure that non-specific activity against human material wasabsent, the antisera were also tested against cell preparationsfrom nasopharyngeal secretions and lungs from which novirus had been isolated.

Patients without respiratory infection could not be used as"controls" because of the absence of sufficient secretion inthe nasopharynx. Figures for virus isolations obtained fromthe examination of cough swabs taken from children withoutrespiratory infection are quoted under Results. Chanock et al.(1958, 1961, 1963) made it clear that R.S. virus andparainflue,nza virus seldom occur in the respiratory tract ofthose without respiratory symptoms.


Results

LABORATORY ASPECTS

During the 22 months from February 1968 to November 197075 children were infected with one or other of theparainfluenza viruses. The first 20 children were diagnosedby culture only, but the results of the neutralization testswere confirmed by immunofluorescence. Nasopharyngealsecretions from the other 55 patients were tested for thepresence of virus by both culture and direct examination byimmunofluorescence. During the same period 133nasopharyngeal secretions were tested for at least one of theparainfluenza viruses by immunofluorescence. Comparison ofthe results of isolation of the virus on monkey kidney cellswith the results of the direct examination of specimens byimmunofluorescence is shown in Table I. The first 20parainfluenza viruses isolated, which are mentioned above,were seven type 1, four type 2, and nine type 3; direct mate-rial for immunofluorescence was not available from thesepatients.

Nasopharyngeal secretions taken from patients from whomparainfluenza viruses were not isolated were examined (Table-II). There were no false-positive results, and the antiserashowed no cross-reaction with the variety of viruses presentin some of these secretions.

TABLE i-Direct Examination of Specimens from Patients by Immuno-fluorescence from Whom Parainfluenza Viruses Were Isolated. September 1969to October 1970

Type of Parainfluenza Virus Total No.Isolated

Immunofluorescent Result

Positive Negative

1 25 23 22 5 5 03 25 25 0

Total .. 55 53 2

Parainfluenza virus type 1: Co-positivity 23/25 (92%), co-negativity 128/128 (100%),overall agreement 151/153 (98-7°h).Parainfluenza virus type 2: Co-positivity 5/5 (100%), co-negativity 87/87 (100%).overall agreement 92/92 (100 On).

Parainfluenza virus type 3: Co-positivity 25/25 (100 %), co-negativity 133/133(100%), overall agreement 158/158 (100%).

Combined parainfluenza virus: Co-positivity 53/55 (96 4°0,), co-negativity 348/348(100 %), overall agreement 401/403 (99 5%).Figures for co-negativity obtained from Table II (After Buck and Gart, 1966)

TABLE II-Immunofluorescent Examination of Nasopharyngeal Secretion fromwhich Parainfluenza Viruses Were not Isolated. September 1969 to October 1970

Type of Para-influenza VirusAntisera againstEach Specimen

Tested

123

No.,of Viruses other thanParainfluenza Virus

I Isolated

U,.>

vi

m

:

ta

0

.0

an

13 *7 *2 *4 27 *4 *2 *3 *29 *8 *2 *4 2

.1r.

-I--I313

TotalNegative

byCulture

9970107

Total TestedNegative byCulture and

Immunofluorescencefor

Parainfluenza Virus

12887133

*One patient had a multiple infection with an H-strain rhinovirus, adenovirus type2, and influenza B.

Of the three types of parainfluenza virus, type 1 was themost difficult to recognize in nasopharyngeal secretionsbecause, on occasions, specimens showed only faint intracel-lular, though specific, fluorescence.

FLUORESCENT APPEARANCES

Fluorescence of the antigen in the three types ofparainfluenza viruses in monkey kidney cells is restricted tothe cytoplasm and appears not oily as fine or coarse particlesbut frequently as strands of fluorescence which form areticulum. A monkey kidney cell infected with parainfluenza

--1-

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virus type 1, examined 36 hours after inoculation with anasopharyngeal secretion, is shown in Fig. 1.

In nasopharyngeal secretions most cells showing fluores-cence are ciliated columnar epithelial cells. The fluorescenceis always cytoplasmic but varies in form in different speci-mens. In the few nasopharyngeal secretions infected with

9

parainfluenza virus type 2 fluorescence is very similar to thatshown by R.S. virus, with fine particles and large spherical oroval inclusion-like bodies (Fig. 2). Parainfluenza virus type 1often produces strands of fluorescence (Fig. 3) and coarseaggregates, but some specimens have also shown cells withfine particles and large inclusions. Parainfluenza virus type 3often produces the particulate type of fluorescence seen withR.S. virus and parainfluenza virus type 2, but a number ofsecretions have shown coarse aggregates and a few haveshown strands of fluorescence in some cells (Fig. 4). Thenasopharyngeal secretion cells infected with parainfluenzavirus type 3 illustrated in Fig. 4 show coarse aggregates andfine particles of virus antigen fluorescence. In the prepara-tions of cells from the tracheal and bronchial secretions of apatient who died, and from whom parainfluenza virus type 1was isolated, large numbers of ciliated epithelial cells werefound containing one or more very large spherical, oval, orirregular inclusion-like bodies, all of which fluoresced withvarying degrees of intensity. In some cells fine particles weredistributed throughout the cytoplasm. The fluorescence wasstrongest in the cells from the trachea (Fig. 5). Necropsyappearance was that of an acute laryngotracheobronchitiswith no microscopical or gross abnormality of the lungs.

FIG. 1-Monkey kidney cell showing cytoplasmic fluorescence of para-influenza virus type 1 antigen 36 hours after inoculation with a specimen ofnasopharyngeal secretion. (x 2,710).

FIG. 4-Parainfluenza virus type 3 antigen in cells of a nasopharyngealsecretion showing cytoplasmic fluorescence in the form of coarse aggregates,fine particles, and occasional strands. ( x 1,735).

FIG. 2-Parainfluenza virus type 2 antigen in two ciliated epithelial cells in anasopharyngeal secretion showing fine particles and larger inclusion-likebodies fluorescing in the cytoplasm. ( x 2,400).

FIG. 3-Parainfluenza virus type 1 antigen in a group of ciliated epithelialcells in nasopharyngeal secretion showing cytoplasmic fluorescence instranded. form ( x 1,870).

FIG. 5-Parainfluenza virus type 1 antigen in ciliated epithelial cells in post-mortem specimen of tracheal secretion showing bright cytoplasmic fluo-rescence in the form of large spherical, oval, or elongated inclusion-likebodies and small particles. ( x 1,735).

Cells eluted from cough swabs and stained by theimmunofluorescent technique for the three parainfluenzaviruses can be used in diagnosis. The appearance ofparainfluenza virus types 1 and 3 in cough swab preparationsis identical with that seen in nasopharyngeal secretions. So farsix out of six cough swabs for parainfluenza virus type 3 andone out of three cough swabs for parainfluenza virus type 1have been successfully diagnosed by direct immunofluores-cence on eluted cells.

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TABLE iII-Comparison between the Haemadsorption Neutralization Tests andthe Fluorescent Antibodcv Technique on Monkey Kidney Cells

Result of Fluorescent Antibody andTvne of Neutralization Tests on Monkey Kidney Cells

ParainfluenzaVirus No.e Iesoted Whioth Neutralization Fluorescent AntibodyWere Tested by Both

Techniques Tests Technique

1 26 26 262 9 9 93 25 25 25

60 60 60

A comparison of the results of immunofluorescence with thestandard haemadsorption neutralization tests (Table III)shows that there was complete agreement between the twomethods. It was decided to limit the comparison to about 25for each type of virus, by which time the reliability of thefluorescent antibody technique would either be proved or

disproved for use as a routine test.


viruses the difference was again highly significant (x2 16-5;P<0.005).To place these findings in perspective the viruses isolated

over one year of the same period from 304 children withacute lower respiratory tract infection but without croup arereported. There were 124 R.S. viruses (40-8%t'h ), 13parainfluenza viruses (4-30)), 12 adenoviruses (4 ,, ), 8rhinoviruses (2-6 /(/), 6 influenza viruses (2',',), 5 herpesviruses(1.6'), and 4 echo and coxsackieviruses (1.3'y,). Duringinvestigations of acute respiratory infections in Newcastlecough swabs were taken from 109 c!hildren without respiratoryinfection and were used as "controls." From them five adeno-viruses, two herpesviruses, and three Coxsackieviruses wereisolated. Neither R.S. virus nor parainfluenza viruses wereisolated from this group.

It has already been noted that parainfluenza virus type 3 isoften associated with lower respiratory tract infection. It wasconsidered important to compare the age distribution of lowerrespiratory tract infection caused by this virus with R.S. virusand parainfluenza virus types 1 and 2. This is given in Fig. 6,

CLINICAL ASPECTS

Parainfluenza viruses are associated with about 8% of allacute respiratory infections in children admitted to hospital.Their relative importance is shown in Table IV, which alsoconfirms the strong association of parainfluenza viruses withcroup, and shows that parainfluenza virus type 3 is far moreoften associated with lower respiratory tract infections thantypes 1 and 2.

TABLE iv-Association between Clinical Category and Parainfluenza VirusIsolation

Parainfluenza Virus % AssociatedClinical Total No. of withCategory Children Type Type Type Parainfluenza

Testeds 1 2 3 Virus

Pneumonia .. 189 2 0 6 4 2Bronchiolitis .. 227 1 0 10 4-8Bronchitis .. .. 262 4 0 7 4-2Croup .. .. 94 18 10 5 351Upper respiratory

tract infection .. 154 4 0 7 7-1

Total .. .. 926 29 10 35 8 0

TABLE V- Viruses Associated with 94 Cases of Croup

Virus No. of Cases % of Total

Parainfluenza 1 .18 19.1Parainfluenza 2 .10 10-6'Parainfluenza 3 .5 5-3R.S. virus 10* 10 6Rhinovirus H .2 2-1Rhinovirus M .1 1.1Adenovirus 6 .1 1.1Influenza A .3 3-2Coxsackie B4 .1 1-1Echovirus type 21 1 1 1No virus isolated 42 44.7

Total ... .. 94 100 0

The association of all viruses, including the parainfluenzaviruses, with the clinical condition of croup is shown in TableV. Though parainfluenza viruses are the main causal agents,R.S. virus plays a significant part. The severity of illness incroup varies from mild to severe, depending on the degreeand speed of development of laryngeal obstruction. It maywell be an important observation that out of 33 patientsinfected with parainfluenza virus croup was consideredsevere in 28 (84%), whereas in 51 children with croup fromwhom parainfluenza virus was not isolated the illness wassevere in only 22 (44%) (x2=9-7; P<0.005). When patientswith croup, and infected with parainfluenza virus, were com-

pared for severity of illness with those infected with other

70-

0

vbIV40-

> 300

0

01

yearsAqe

FIG. 6-Age incidence of R.S. virus and parainfluenzavirus infection in children. February 1968 to October 1970.

which shows the similar age distribution of parainfluenzavirus type 3 and R.S. virus and contrasts sharply with thatfor parainfluenza virus types 1 and 2. The small number ofpatients between 1 and 4 years of age infected withparainfluenza virus type 3 are those with croup.

Discussion

An effective and reliable method for the rapid diagnosis ofparainfluenza virus infections by immunofluorescence within24 hours of the patient's admission to hospital has beenestablished by using cells from nasopharyngeal secretions; co-

positivity and co-negativity estimates have confirmed thereliability of the test (Buck and Gart, 1966). Cells eluted fromcough swabs could also be stained, but this method appearedto be less sensitive. The conventional method for the isolationof parainfluenza virus is on primary or secondary monkeykidney cells, the haemadsorption test for detection and thehaemadsorption neutralization test for identification beingused. Monkey kidney cells are not always available and are

occasionally contaminated with simian haemadsorptionviruses (SV5 and SV41), which makes detection of humanparainfluenza viruses difficult. Furthermore, parainfluenzaviruses in specimens are not easy to store, and attempts to

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reisolate a virus from a stored specimen often fail. For thesereasons, too, the use of immunofluorescence for the diagnosisof infection with parainfluenza virus has proved valuable.

It is now possible to diagnose by immunofluorescence allthe known major virus pathogens causing acute respiratoryinfections in children, and a technique applicable to all of themis now available (Gardner and McQuillin, 1968; McQuillinand Gardner, 1968; McQuillin et al., 1970). Immunofluores-cence for the rapid diagnosis of acute respiratory infectionmust be used with discretion. To make it economic, factorssuch as local prevalence of a virus, the clinical condition,season of year, and age of patient must be considered.For example, R.S. virus infects children mainly under the ageof 1 year; the most frequent cause of bronchiolitis is R.S.virus, and though parainfluenza virus infections by types 1and 3 may occur in epidemics they are less frequent thanthose caused by R.S. virus and less often restricted to winter.With such discrimination immunofluorescence can become aninvaluable technique in diagnostic virology.

Immunofluorescence has also proved reliable for the detec-tion of parainfluenza viruses in monkey kidney tissue culture.We have found no significant cross-reaction between the threetypes investigated on monkey kidney cells and a minimalcross-reaction between parainfluenza virus type 1 antiserumand parainfluenza type 3 virus in cells of nasopharyngealsecretions; therefore, both antisera should be used in parallel.So far there are no reports by other workers of direct exami-nation of nasopharyngeal secretions by immunofluorescencefor parainfluenza viruses. Chin (1963) and Fedova andZelenkova (1969) found some cross-reaction between theparainfluenza virus types and other haemadsorption viruses onmonkey kidney cells. The identification of a haemadsorptionvirus is time-consuming when the haemadsorption neutral-ization test is used, but the same identification can be made aseffectively in a few hours by immunofluorescence. In routinevirology a frequent problem is the identification of a haem-adsorption virus on monkey kidney cells.The following haemadsorption viruses can now be identified

immediately: parainfluenza virus types 1, 2, and 3, and influ-enza virus A and B. These are the most likely pathogens togive a positive haemadsorption reaction. They show no cross-reaction by immunofluorescence with influenza C, mumps, orSV5, which are also haemadsorption viruses. We have noinformation about their cross-reaction with parainfluenzavirus types 4a and 4b. Mumps and influenza C can be iden-tified by a complement fixation test and SV5 withparainfluenza virus types 4a and 4b by a haemadsorption neu-tralization test. This system has simplified the identification ofa haemadsorption virus.

Variation in the types of cytoplasmic fluorescence is seenwith different parainfluenza viruses in nasopharyngeal cellsfrom patients. No significance can be attached to these find-ings without the investigation of a larger number ofparainfluenza virus infections and having available the precisedetails of the stage of illness at the time when nasopharyngealspecimens are examined. Differences in the appearance offluorescence of parainfluenza virus antigens in various tissueculture cells have been described by many workers, with ref-erence to the development of the type of antigen (S soluble orV virus particle) in different sites of the cell, according to thestage of infection (Zhdanov et al., 1965; Howe et al., 1967;Sominina et al., 1967; Fedova' and Zelenkova, 1969).The cause of the weak fluorescence occasionally observed

in the cells of nasopharyngeal secretions in parainfluenzavirus type 1 infections has yet to be determined. It may bedue to an antigenic difference between the infecting strainand the strain of parainfluenza virus type 1 (Sendai) used toprepare the antiserum. Alternatively, it could be due to a

"blocking" of staining by local antibody in a later stage of ill-ness, similar to that observed in R.S. virus infections(Gardner et al., 1970).

11

Parainfluenza viruses may also be associated with fatal res-piratory illness. Parainfluenza virus type 3 was foundassociated with pneumonia in an infant death (Aherne et al.,1970), and in the present series parainfluenza virus type 1was associated with death in an infant with acute laryngo-tracheobronchitis. In this second child virus was found byboth immunofluorescence and isolation techniques in thenasopharyngeal secretions before death and in tracheal andbronchial secretions after death, but not in the lungs. Thiswas in agreement with the necropsy findings when the lungsappeared normal, both macroscopically and microscopically.Parainfluenza virus type 1 variety Sendai was first describedas a cause of death in the newborn by Sano et al. (1953) andKuroyo et al. (1953), but the relationship of virus to illnesswas obscured by the isolation of virus in mice, known onoccasions to carry this virus.

CLINICAL IMPORTANCE

Chanock et al. (1963) first showed the clinical importance ofthese three parainfluenza viruses in a five-year investigationof children admitted to hospital with acute respiratory infec-tions and comparing them with controls. They found that 29%of croup cases was caused by these viruses and also a smallbut significant number of acute lower respiratory infections.Parainfluenza viruses were also associated with 6 0/, of allacute respiratory infections; a similar incidence was found byVan der Veen Smeur (1961) in Holland. The results obtainedin the present study are similar to those previously recorded,but the importance of parainfluenza virus types 1 and 2 ascauses of croup is noteworthy; parainfluenza virus type 2 wasnot associated with any other acute respiratory syndrome.The severity of croup also appeared to be related to thecausal organism, the illness being more severe in patientsinfected with parainfluenza viruses than in those infectedwith other viruses or in those in whom no virus was isolated.Parainfluenza virus type 3 and, to a smaller extent, type 1caused acute lower respiratory infections.The clinical features of acute lower respiratory tract infec-

tions caused by parainfluenza virus type 3 are similar tothose associated with R.S. virus infections. A comparison wastherefore made of the age distribution of parainfluenza virustype 3 infections with those caused by R.S. virus (Fig. 6).They both caused severe lower respiratory infections in thoseunder 6 months, which was not a feature of parainfluenzavirus type 1 and 2 infections. All three parainfluenza virusesand R.S. virus may cause croup in those over 1 year of age.Chanock et al. (1970) speculated that a type 3 allergic reac-

tion (Gell and Coombs, 1968) involving the formation of toxiccomplexes by virus and circulating maternal IgG may be afactor in the pathogenesis of severe R.S. virus infections ofinfancy. Gardner et al. (1970), though not ruling out a type 3reaction, suggested a type 1 reaction involving sensitizationby previous exposure to the ubiquitous R.S. virus as a morelikely explanation. Though severe lower respiratory infectionsof infancy may have similar pathogenesis in the two virusinfections, parainfluenza virus type 3 epidemics are lessfrequent than those due to R.S. virus. This may account forminor differences in the percentage distribution of the twoinfections in older children (Fig. 6).

This communication has shown the position of theparainfluenza viruses as pathogenic agents in acute respira-tory infections in children and the techniques which can bereliably used in their rapid diagnosis and identification. Thesimilarity in the clinical behaviour of parainfluenza virus type3 and R.S. virus suggests the need for the further investiga-tion of the pathogenesis of illness associated with these twoviruses.

We are indebted to Professor S. D. M. Court and his colleaguesin the department of child health and to the ward staff for their co-

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operation. We are also grateful to the technicians in the depart-ment of virology for their help. Thanks are also due to the Scien-tific and Research Subcommittee of the United Newcastle uponTyne Hospitals and the Medical Research Council for financialsupport.

ReferencesAherne, W., Bird, T., Court, S. D. M., Gardner, P. S., and McQuillin, J.

(1970). Journal of Clinical Pathology, 23, 7.Banatvala, J. E., Anderson, T. B., and Reiss, B. B. (1964). British Medical

journal, 1, 537.Buck, A. A., and Gart, J. J. (1966). American Jfournal of Epidemiology,

83, 586.Chanock, R. M., et al. (1958). New England journal of Medicine, 258, 207.Chanock, R. M., et al. (1961). Journal of the American Medical Association,

176, 647.Chanock, R M., Parrott, R. H., Johnson, K. M., Kapikian, A. Z., and Bell,

J. A. (1963). American Review of Respiratory Diseases, 88, Suppl., p. 152.Chanock, R. M., Kapikian, A. Z., Mills, J., Kim, H. W., and Parrott, R. H.

(1970). Archives of Environmental Health, 21, 347.Chin, T. D. Y. (1963). American Review of Respiratory Diseases, 88, Suppl.,

p. 334.Fedovd, D., and Zelenkovi, L. (1969). J'ournal of Hygiene, Epidemiology,

Microbiology and Immunology, 13, 13.

Gardner, P. S. (1968). Archives of Disease in Childhood, 43, 629.Gardner, P. S., and McQuillin, J. (1968). British Medical Journal, 3, 340.Gardner, P. S., Stanfield, J. P., Wright, A. E., Court, S. D. M., and Green,

C. A. (1960). British Medical Journal, 1, 1077.Gardner, P. S., McQuillin, J., and Court, S. D. M. (1970). British Medical

Journal, 1, 327.Gell, P. G. H., and Coombs, R. R. A. (1968). Clinical Aspects of Immunology,

2nd edn. Oxford, Blackwell Scientific.Holzel, A., et al. (1963). Lancet, 1, 295.Holzel, A., et al. (1965). British Medical Journal, 1, 614.Howe, C., Morgan, C., De Vaux St. Cyr, C., Hsu, K. C., and Rose, H. M.

(1967). Journal of Virology, 1, 215.Kuroyo, M., Ischida, N., and Shiratori, T. (1953). Yokohama Medical

Bulletin, 4, 217.McQuillin, J., and Gardner, P. S. (1968). British Medical Journal, 1, 602.McQuillin, J., Gardner, P. S., and McGuckin, R. (1970). Lancet, 2, 690.Sano, T., Niitsu, I., Nakagawa, I., and Ando, T. (1953). Yokohama Medical

Bulletin, 4, 199.Sominina, A. A., Zubzhitsky, Y. N., and Smorodinstev, A. A. (1967). Acta

Virologica, 11, 424.Sturdy, P., McQuillin, J., and Gardner, P. S. (1969). Journal of Hygiene,

67, 659.Van der Veen, J., and Smeur, F. A. A. M. (1961). American Journal of

Hygiene, 74, 326.Vogel, J., and Shelokov, A. (1957). Science, 126, 358.Zhdanov, V. M., Azadova, N. B., and Uryvayev, L. V. (1965). Journal of

Immunology, 94, 658.

Coagulation and Fibrinolytic Systems in Pre-eclampsiaand EclampsiaJOHN BONNAR, G. P. McNICOL, A. S. DOUGLAS

British Medical Journal, 1971, 2, 12-16

Summary

The coagulation and fibrinolytic mechanisms were investigatedin a group of patients with severe pre-eclampsia and eclampsiaand the findings were compared with those of healthy womenin late pregnancy. In patients with pre-eclampsia the followingsignificant differences were found: (1) greater depression ofplasma fibrinolytic activity (euglobulin lysis time) than in nor-mal pregnancy, (2) a higher level of inhibitor to urokinase-induced lysis, (3) increased levels of serum fibrin degradationproducts, and (4) reduced platelet counts.

In patients with eclampsia a progressive increase of thelevel of serum fibrin degradation products was found over thethree days following eclamptic seizures. No such increaseoccurred after grand mal seizures in late pregnancy. The find-ings in this study support the view that intravascular clottingis taking place in pre-eclampsia and that this disturbance ofthe balance between coagulation and fibrinolysis may belocalized to certain areas of the vascular compartment, partic-ularly the placental and renal circulations. Fibrin deposition inthe maternal vessels supplying the placenta would impair theplacental blood flow, which may explain the placental insuf-ficiency which occurs in pre-eclampsia. Likewise fibrin depo-sition in the renal vasculature will result in glomerular dam-age and proteinuria. Hypertension may be related to the renal

Nuffield Department of Obstetrics and Gynaecology, Radcliffe Infirmaryand Churchill Hospital, Oxford OX3 7LJ

JOHN BONNAR, M.D., M.R.C.O.G., First Assistant

University Department of Medicine, Royal Infirmay, Glasgow C.4G. P. McNICOL, F.R.C.P., Reader in MedicineA. S. DOUGLAS, F.R.C.P., Professor of Medicine (Present appointment:

Regius Professor of Medicine, University of Aberdeen)

ischaemic changes or a compensatory response to the presenceof fibrin deposition in the vascular compartment. This evi-dence of intravascular fibrin deposition raises the question ofthe possible therapeutic value of antithrombotic agents to in-hibit the clotting process. On a theoretical basis such treat-ment might be expected to improve blood flow to the placentaand thereby fetal growth.

Introduction

The aetiology of pre-eclampsia remains obscure. Jeffcoate(1966) summarized many of the pathological factors known tobe associated with pre-eclampsia and affirmed that it was adisease of theories; the possible role of the coagulation andfibrinolytic systems was not included and this aspect hasreceived little attention. In some fatal cases of eclampsia,however, a prominent finding has been widespread fibrindeposition (McKay et al., 1953). Electron microscopical studyof tissue obtained by renal biopsy from patients with pre-eclampsia has revealed swelling of the glomerular capillaryendothelium and deposition of an amorphous fibrinoidmaterial within the cells and beneath the basement membrane(Pollak and Nettles, 1960; Altchek, 1961). Morris et al. (1964)using immunofluorescent techniques showed that the materialin the glomeruli was identical to fibrin.

Recently evidence has been accumulating that the fibrinolyticsystem may be implicated in the mechanisms which influenceblood pressure and blood flow (Niewiarowski, 1968). Thephysiological role of plasminogen activator is most likely tokeep the blood vessels free of fibrin deposits, and in situa-tions where the secretion or action of plasminogen activator isimpaired fibrin deposition may be more likely to occur. Toour knowledge there have been no detailed and serial studiesof components of the fibrinolytic enzyme system in a well-defined group of patients with severe pre-eclampsia andeclampsia. The following investigation was undertaken to

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observations clinical and immunofluorescent diagnosis

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